AIM: To investigate the role of caveolin-3 (CAV3) and cholecystokinin A receptor (CCKAR) in cholesterol gallstone disease (CGD).

METHODS: To establish a mouse model of CGD, male C57BL/6 mice were fed with a lithogenic diet containing 1.0% cholic acid, 1.25% cholesterol and 15% fat; a similar control group was given a normal diet. The fresh liver weights and liver-to-body weight ratio were compared between the two groups after one month. Serum lipid profile and bile composition were determined with an autoanalyzer. The Cav3 and Cckar mRNA and CAV3 and CCKAR protein levels in the liver and gallbladder were determined via real-time polymerase chain reaction and Western blot, respectively.

RESULTS: Establishment of the mouse CGD model was verified by the presence of cholesterol gallstones in mice fed the lithogenic diet. Compared with mice maintained on a normal diet, those fed the lithogenic diet had significantly higher mean liver-to-body weight ratio (0.067 ± 0.007 vs 0.039 ± 0.007, P < 0.01), serum total cholesterol (4.22 ± 0.46 mmol/L vs 2.21 ± 0.11 mmol/L, P < 0.001), bile total cholesterol (1.33 ± 0.33 mmol/L vs 0.21 ± 0.11 mmol/L, P < 0.001), and bile phospholipid concentrations (3.55 ± 1.40 mmol/L vs 1.55 ± 0.63 mmol/L, P = 0.04), but lower total bile acid concentrations (726.48 ± 51.83 μmol/L vs 839.83 ± 23.74 μmol/L, P = 0.007). The lithogenic diet was also associated with significantly lower CAV3 in the liver and lower CAV3 and CCKAR in the gallbladder compared with the control mice (all P < 0.05).

CONCLUSION: CAV3 and CCKAR may be involved in cholesterol gallstone disease.

Cholesterol gallstone disease (CGD) accounts for 80%-90% of the gallstones found at cholecystectomy, and is the most common digestive disease requiring hospital admission[1,2]. Epidemiological studies conducted in the United States showed that 10%-15% of adults have CGD, and more than one million people are newly diagnosed annually[3,4]. The prevalence of CGD has increased in developing countries possibly due to the western lifestyle adopted[5,6].

CGD results from bile lipids and bile salts imbalance in the gallbladder[7]. The development of CGD is associated with supersaturation of bile with cholesterol, rapid precipitation of cholesterol crystals in the gallbladder, increased bile salt hydrophobicity, and inflammation of the gallbladder[8]. Of these events, precipitation of excess cholesterol in the bile is a prerequisite for gallstone formation[9]. In general, cholesterol is solubilized in mixed micelles together with bile salts and phospholipids in bile. However, cholesterol precipitation and subsequent cholesterol gallstone formation may occur as a result of cholesterol supersaturation in bile[10,11].

Multifactorial factors are involved in the mechanism of CGD[12]. Impaired gallbladder emptying, which provides sufficient time for cholesterol crystal nucleation, contributes significantly to gallstone formation[13]. Caveolins are a family of small integral membrane proteins consisting of caveolin-1, caveolin-2, and caveolin-3 (CAV3)[14]. Caveolin-1 and caveolin-2 are the major structural proteins of caveolae, and are abundantly coexpressed in endothelial and adipose cells[15], while CAV3 is mainly found in muscle cells[16]. Caveolins are postulated to be mainly involved in modulation of cholesterol movement and storage[14,17]. A recent in vitro study observed that CAV3 was involved in the regulation of gallbladder muscle hypomotility[18]. Knockdown of CAV3, mediated via small interfering RNA, decreased contraction of gallbladder muscle cells isolated from guinea pigs, and increased cholecystokinin A receptor (CCKAR) in the caveolae[18]. CCKAR is a major physiologic mediator of smooth muscle contraction of the gallbladder. Lack of CCKAR may deteriorate gallbladder contraction and enhance gallstone formation[19]. Polymorphism of Cckar gene was associated with gallstone formation in humans[20,21].

To date, the in vivo association between CAV3 and CGD has not been fully elucidated, and whether CCKAR is differentially expressed during the process of cholesterol gallstone formation remains uncertain. In this study, we established a mouse model of CGD induced by a lithogenic diet, and investigated the association of CAV3 and CCKAR with CGD.

Mice fed with lithogenic diet containing 1.0% cholic acid, 1.25% cholesterol and 15% fat for one month developed gallstones. In addition, the mice fed the lithogenic diet had significantly higher mean liver weight and liver-to-body weight ratio compared with the control mice fed a normal diet (Figure 1). Histological analysis showed significant fatty infiltration of the liver in mice fed a lithogenic diet, while the control mice exhibited normal liver architecture.

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Figure 1 Liver-to-body weight ratio in mice fed a normal or lithogenic diet.

Mice fed a lithogenic diet had significantly higher levels of serum total cholesterol, HDL-C, and LDL-C compared with the control mice (Table 2). Bile total cholesterol and phospholipid concentrations were also significantly higher in mice fed a lithogenic diet, while total bile acid levels in bile were significantly lower (Table 2).

To investigate the associations between CAV3 and CCKAR and CGD, the mRNA levels of Cav3 and Cckar in the livers and gallbladders were analyzed. Real-time PCR showed that mRNA levels of Cav3 and Cckar in the gallbladder of mice fed a lithogenic diet were significantly lower than those of the control mice (Figure 2A). Cav3 mRNA levels in the liver of mice fed a lithogenic diet was also significantly lower than those of the controls (Figure 2B). Cckar mRNA was not detected in the liver.

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Figure 2 Cav3 and Cckar mRNA in the gallbladder (A) and liver (B) of both control and cholesterol gallstone disease mice.

The proteins CAV3 and CCKAR were analyzed in the livers and gallbladders by Western blot. According to the mRNA expression results, the protein levels for CAV3 and CCKAR in the gallbladder of mice fed a lithogenic diet were significantly lower compared with the controls (Figure 3). The protein levels of CAV3 in the liver of mice fed a lithogenic diet were also significantly lower than in the controls (Figure 4). These results suggested that the development of CGD was accompanied by downregulation of CAV3 expression in the liver, and downregulation of CAV3 and CCKAR expression in the gallbladder, implying that CAV3 and CCKAR may be involved in CGD.

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Figure 3 Western blotting for caveolin-3 and cholecystokinin A receptor in the gallbladder of both control and cholesterol gallstone disease mice. CAV3: Caveolin-3; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

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Figure 4 Western blotting for caveolin-3 in the liver of control and cholesterol gallstone disease mice. CCKAR: Cholecystokinin A receptor; CAV3: Caveolin-3; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

In this study, we established a mouse model of CGD and observed that the formation of gallstones was accompanied by increases in serum and bile total cholesterol concentrations, while total bile acid concentrations in bile decreased. Lower levels of hepatic CAV3 expression, and CAV3 and CCKAR in the gallbladder were also found in the mouse model compared with the control mice.

The mechanisms underlying the pathogenesis of CGD are incompletely understood[22]. Epidemiologic studies imply the involvement of multiple environmental factors and genetic elements in cholesterol gallstone formation[23,24]. The former includes diet as a risk factor for cholesterol gallstone formation[25]. In the present study, mice developed cholesterol gallstones fed for one month with a lithogenic diet containing 1.0% cholic acid, 1.25% cholesterol and 15% fat. Significant increases in serum total cholesterol and LDL-C levels were observed in mice fed the lithogenic diet. This change mimics the alterations in serum lipid profiles observed in CGD patients[26,27].

Hypersecretion of biliary cholesterol and cholesterol supersaturation of the bile are the most important prerequisites for gallstone formation[11]. Bile acids are the major components of bile, and are involved in cholesterol elimination. Decreases in total bile acid levels may be associated with reduced cholesterol elimination in the bile and subsequent cholesterol supersaturation. In the present study, we observed that bile total cholesterol and phospholipid levels were significantly higher in the mouse CGD model, while total bile acid concentrations in bile were lower than in the control mice.

Decreased gallbladder motility is crucial to the formation of cholesterol crystals in bile[13]. The mechanisms associated with decreased gallbladder motility remain unclear. Caveolins are scaffolding proteins that have important roles in cholesterol homeostasis[14,17]. Besides its role in cholesterol metabolism, CAV3 was also found to be involved in regulation of gallbladder muscle hypomotility in vitro[18]. However, the in vivo association between CAV3 and CGD is not fully understood. In this study, we observed that CAV3 expression was lower in the liver and gallbladder of the mouse model of CGD, compared with the normal controls.

CCKAR is a major mediator of smooth muscle contraction of the gallbladder. One-third of CCKAR (-/-) mice spontaneously develop gallstone disease at 12 and 24 mo of age[19]. Polymorphism of Cckar gene in patients is also an independent risk factor for gallstone disease[21]. However, whether CCKAR is differentially expressed during the process of CGD formation remains unclear. Here, we observed that the expression of CCKAR was significantly lower in the gallbladder of CGD mice than in the control group. These observations suggest the involvement of CAV3 and CCKAR in cholesterol gallstones. However, the precise mechanism remains to be determined. Further study is also needed to clarify whether our findings are relevant to humans, and the possibility of therapeutic intervention for the disease.

In conclusion, our results showed that both mRNA and protein of CAV3 and CCKAR were differentially expressed in the liver and gallbladder of a mouse model of CGD compared with control mice. Further investigation may enhance our understanding of the pathogenesis of CGD, and enable exploration of novel therapeutic targets.